Regulatory aspects of the cytoplasmic 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) receptor will be studied in vitamin D-deficient chickens and rats. The proposed experiments will utilize both the intestine and bone as sources for these cytoplasmic receptors. These studies will be directed towards gaining an understanding of the regulation of these receptors by investigating binding to 1,25(OH)2D3 and DNA. (1) We will first establish whether or not physical and/or chemical changes occur to the receptor due to ligand binding and whether the changes (activation) are necessary for DNA binding to occur. These experiments will involve measuring chromatographic and electrophoretic behavior as well as analysis by sucrose density gradients. Alterations in receptor activity will be quantitated by measuring receptor binding to immobilized DNA or synthetic DNA. (2) Since calcium and phosphorous concentrations in vivo are instrumental in regulating the vitamin D endocrine system, we will attempt to ascertain whether these ions are also important in regulating receptro binding of 1,25(OH)2D3 and consequently the 1,25(OH)2D3-receptor complex to DNA. (3) The cytoplasmic 1,25(OH)2D3-receptor complex will be studied in the presence of a variety of immobilized nucleotides (i.e., DNA, poly, d(T), poly d(A), ATP etc.) to gain insight into the specificity and dynamics of this binding interaction. Pyridoxal phosphate and intercalating drugs will be utilized as chemical probes to investigate whether these receptor-DNA binding forces are electrostatic or hydrophobic. (4) Site directed affinity labeling with halomethyl ketones will be used as a means to identify the functional residue (s) for 1,25(OH)2D3 binding while discrimination of the DNA and ligand binding sites on the receptor will be carried out with Cibacron blue.